The polycystic ovarian syndrome (PCOS) represents the most common reproductive pathophysiology in pre-menopausal women. Hallmarks of PCOS are increased LH secretion, altered insulin action, and augment ovarian androgen biosynthesis. Overall working hypothesis is that LH and insulin synergize at the level of the ovarian theca cell to drive excessive androgen secretion. Although LH and insulin ( or IGF-1) can synergistically amplify ovarian androgen biosynthesis in vivo and by human, hen, rat and pig theca cells in vitro (present data), how LH and insulin collaborate in this fashion is not known. This project will investigate the clinical and molecular mechanisms of lH and insulin synergy in PCOS patients in vivo and on (pig) theca cells in vitro. Our individual aims arise from the following specific hypotheses: I. Preferential suppression of hyperinsulinism (via metformin treatment) of LH hypersecretion (via leuprolide down-regulation) alters the testosterone secretory response to human recombinant LH infusions in PCOS patients; II. In vitro, in pig theca-cell populations, LH and insulin synergistically up-regulate the molecular expression of critical genes that control sterol commitment to androgen biosynthesis; namely, the low- density lipoprotein (LDL) receptor, the steroidogenic acute responsive protein (StAR), and the 17-alpha hydroxylase enzyme; III. In situ, at the single-theca-cell level, LH and insulin synergize by coordinately enhancing multiple sterol-regulatory gene co-expression in individual theca cells; and IV. There are pivotal cis-DNA promoter elements in the StAR and LDL receptor genes that mediate responses to the intracellular signals generated by insulin and LH, acting singly and synergistically. The preceding clinical and basic-science hypotheses and corresponding experiments should help identify novel clinical and molecular mechanisms that govern theca-cell androgen biosynthesis, and thereby offer new insights into the basic pathobiology of PCOS.